The present invention relates to the use of recombinant polypeptides and synthetic peptides derived from a horseshoe crab Factor C as well as computationally designed peptide analogues, all of which have endotoxin-binding domain(s). The recombinant proteins may be expressed from insect cell clones, either as is or as fusion proteins, e.g. with green fluorescent protein (GFP). The extreme sensitivity of the present recombinant Factor C to LPS, with its unique LPS-binding domains which have unsurpassed binding affinity for LPS, may be exploited in accordance with the present invention for anti-endotoxin and anti-microbial therapeutics as well as for the tracing, detection, and removal of LPS or gram-negative bacteria. The present invention also relates to a method for treating bacterial infection of a subject by inducing bacteriostasis by administration of a recombinant Factor C protein.
Endotoxin, also known as lipopolysaccharide (LPS), is an integral component of the gram-negative bacterial cell membrane and is responsible for many, if not all, of the toxic effects that occur during gram-negative bacterial sepsis (1). LPS is a mixture of glycolipids consisting of a variable polysaccharide domain covalently bound to a conserved glucosamine-based phospholipid known as lipid A. LPS directly stimulates host monocytes and macrophages to secrete a wide array of inflammatory cytokines that include tumor necrosis factor-xcex1 (TNF-xcex1), interleukins-1 (IL-1), and interleukin-8 (IL-8) (2). Excessive release of these cytokines by host macrophages almost assuredly contributes to organ failure and death that occur after episodes of gram-negative bacterial sepsis (3). The proinflammatory bioactivities exhibited by LPS typically reside in the lipid A moiety (4).
LPS from gram-negative bacteria induces the amoebocytes of horseshoe crabs to aggregate and degranulate. Presumably, the LPS-induced coagulation cascade represents an important defense mechanism used by horseshoe crabs against invasion by gram-negative bacteria (5). The amoebocyte lysate constituted as the Limulus amoebocyte lysate (LAL) test has been used for decades as a tool for detecting trace concentrations of LPS in solution (6,7). The molecular mechanism of coagulation in horseshoe crab has been established and it involves a protease cascade. This cascade is based on 3 kinds of serine protease zymogens, Factor C, Factor B, proclotting enzyme, and one clottable protein, coagulogen (8). Being the initial activator of the clotting cascade, Factor C functions as a biosensor that responds to LPS.
Despite advances in antimicrobial therapy, septic shock and other clinical complications due to Gram-negative bacterial infections continue to pose a major problem. Endotoxin or lipopolysaccharide (LPS) present on the cell wall of Gram-negative bacteria (GNB) plays an important role in the pathophysiology of these infections. It does so by mediating toxicity and also mediating release of factors like tumor necrosis factor and interleukins (40), and also by forming a rigid shield around the bacteria protecting them from the effects of antibiotics. Therefore, the detection and/or removal of LPS from the bloodstream or any parenteral solution may aid in the prevention of the inflammatory and pyrogenic effects of LPS. The lipid A component of LPS plays the most important biological role; lipid A gives rise to all the ill effects elicited by endotoxin.
A number of LPS-binding proteins have been identified. Among them are the LPS binding protein, LBP (41), and bactericidal permeability increasing protein, BPI (18,42). LBP, a 60 kDa mammalian serum protein, has a binding site with a high degree of specificity for lipid A (43). BPI, a 55 kDa protein found in human neutrophils, is capable of binding to the toxic lipid A moiety of LPS resulting in neutralization of the endotoxin (18,42,44,45).
The circulating amoebocytes of the horseshoe crab contain an array of proteins that are capable of binding and neutralizing LPS. The Limulus antilipopolysaccharide factor, LALF, an 11.8 kDa LPS-binding peptide, has been identified in the amebocytes of horseshoe crabs Limulus polyhemus and Tachypleus tridentatus. LALF has subsequently been isolated and characterized (46-49). Purified LALF has been shown to bind LPS and exhibit endotoxin neutralization (50,19,51,52). Two other LPS-binding proteins from horseshoe crab hemocytes are tachyplesin (53,54) and big defensin (55).
Factor C is a serine protease zymogen. It is the key enzyme in the C. rotundicauda amoebocyte lysate (CAL) that is activated by LPS to initiate the coagulation cascade (56-58). Factor C activity is the basis of a very sensitive assay for femtogram levels of endotoxin used in the quality control of pharmaceutical products (59). The importance of Factor C in the detection of endotoxin has thus led to the expression of recombinant Factor C, rFC (12,60,61,73-38), as an alternative source that should alleviate the batch-to-batch and seasonal variation in the sensitivity of detection of endotoxin which is a recognized drawback with conventional amoebocyte lysate (59-61).
Since Factor C can be activated by femtograms of LPS, it is thought that Factor C has an LPS-binding region that exhibits exceptionally high affinity for LPS. Consequently, this LPS-binding domain can be utilized to detect and remove pyrogenic contaminants in pharmaceutical products intended for parenteral administration as well as for in vivo immunohistochemical determination of endotoxin localization (9).
The LPS-binding property of Factor C resides in the amino-terminal regions spanning 333 amino acids. This short region constitutes a signal peptide, a cysteine-rich region, followed by epidermal growth factor-like domain and finally 3 sushi domains. High LPS affinity, comparable to the native Factor C, requires the correct formation of 9 disulfide bonds (16). This obstacle is compounded by the presence of a cysteine-rich region. Here, for the first time, we report the expression and secretion of a functional LPS-binding domain of C. rotundicauda Factor C (SSCrFCES) via a novel secretory signal. The secretory signal (SEQ ID NO: 17) is disclosed in U.S. patent application Ser. No. 09/426,776, filed Oct. 26, 1999. The entire disclosures of Ser. No. 09/426,776 and of the provisional application upon which it is based, Ser. No. 60/106,426, are hereby expressly incorporated by reference.
Homologous Factor C zymogen cDNAs have been cloned from one of the four extant species of horseshoe crab, Carcinoscorpius rotundicauda (CrFC) (10). Initial attempts to express CrFC and its truncated forms in E. coli resulted in a non-active enzyme (11). Subsequently, CrFC was cloned and expressed in Saccharomyces cerevisiae and a methylotropic yeast, Pichia pastoris. However, neither the Factor C nor the Saccharomyces cerevisiae a mating factor signal sequences were capable of directing secretion of the recombinant protein into the culture media for easier purification (12). Full-length CrFC expressed in yeast was not enzymatically active although it retained endotoxin-binding properties (13).
Expression in a baculoviral system (U.S. patent application Ser. No. 09/081,767, filed May 21, 1998) yielded recombinant Factor C (rFC) with LPS-inducible enzyme activity. The entire disclosures of Ser. No. 09/081,767 and of the provisional application upon which it is based, Ser. No. 60/058,816, are hereby expressly incorporated by reference. The rFC has extremely high sensitivity to trace levels of LPS ( less than 0.005 EU/ml). Before these experiments, the LPS-binding domain of Factor C exhibiting high affinity for LPS was never before successfully expressed in a heterologous host. The difficulty in doing so was largely due to its highly complex mosaic structure. While many highly disulfide-bonded proteins, like epidermal growth factor (14) and secreted acetylcholinesterase (15), were successfully expressed, few display the kind of complexity posed by the Factor C LPS-binding domain.
A form of SSCrFCES was secreted in accordance with the present invention and was purified to homogeneity. The biological functions of the recombinant SSCrFCES were assessed by measuring the ability of the SSCrFCES to bind lipid A using an ELISA-based lipid A binding assay as well as surface plasmon resonance interaction. Other subfragments containing the LPS-binding domain(s)xe2x80x94e.g., SSCrFCsushi-1,2,3-GFP, SSCrFCsushi-1-GFP, SSCrFCsushi-3-GFP (fusion constructs with green fluorescent protein, GFP)xe2x80x94as well as synthetic peptides, e.g., sushi-1 (S1), sushi-1xcex94 (S1xcex94), sushi-3xcex94 (S3), and sushi-3xcex94 (S3xcex94), each of 34 mer length, and designed variant forms of peptides bearing BHBHB and/or BHPHB (where B=basic, H=hydrophobic, P=polar amino acids)xe2x80x94also show strong affinity for endotoxin.
The ability of these proteins and peptides to mediate inhibition of endotoxin-induced Limulus amoebocyte lysate (LAL) coagulation was measured with a sensitive LAL Kinetic-QCL assay. The SSCrFCES protein and peptides were also tested for their ability to suppress LPS-induced cytokines (TNF-xcex1 and IL-8) produced by THP-1 and normal human peripheral blood mononuclear cells (hPBMC). SSCrFCES and the peptides were non-cytotoxic. SSCrFCES has a clearance rate of 4.7 m/min. We also show that low doses of SSCrFCES protein and the synthetic peptides protect galactosamine-sensitized mice from LPS-induced lethality. The peptides have strong antimicrobial potencies and can therefore be used as potent therapeutics.
The present invention thus includes treating bacterial infections by administration of proteins peptides that will bind to endotoxins, especially endotoxins produced by gram-negative bacteria, to an infected subject. The binding is apparently mediated by the lipid A component of the endotoxin. The administered protein induces bacteriostasis (that is, inhibition of bacterial proliferation) in the subject.
A preferred embodiment of this aspect of the invention is one wherein recombinant Factor C is the administered protein. The recombinant Factor C can be a full-length Factor C protein, or any portion thereof that retains the activity of binding to lipid A. It is not necessary that the Factor C retain its serine protease enzymatic activity for the protein to be effective in the method of the invention. It may in fact be beneficial if the serine protease activity is absent.